The long range objective of this research is to determine the structural organization of eukaryotic centromeres and to investigate the molecular mechanisms of chromosome segregation in mitosis and meiosis. The fission yeast, Schizosaccharomyces pombe will be further developed as an experimental system, because this simple eukaryote is amenable to biochemical, genetic, and cytological investigations. The centromere regions of the three S. probe chromosomes are large, include repeated DNA sequences, and,in several ways, are similar to centromeres of higher eukaryotes. Specific aims of the proposed research are to: (1) complete the isolation of centromeric DNA from all three S. pombe chromosomes by a plasmid integration and nucleolytic excision method; (2) characterize this DNA with regard to functional centromere lengths; determine the overall organization and roles of repeats B and K and other centromere-specific repeated DNA sequences with regard to function; and investigate the chromatin structure of S. pombe centromere regions; (3) identify and map RNA transcripts of centromeric DNA sequences; (4) continue to evaluate the minichromosome system as an assay for centromere function in S. pombe; develop genomic substitution vectors for assaying centromere function directly in parental chromosomes of diploid strains; and examine the behavior of various altered cen structures through nuclear divisions; (5) isolate and characterize proteins that interact specifically with centromeres using biochemical and genetic methods; identify specific protein binding sites on centromere DNA; (6) compare centromeres of S. pombe, S. cerevisiae, and Aspergillus nidulans with regard to sequence homologies, centromere-interacting proteins, and cross-species function. The investigation of the molecular mechanisms of chromosome segregation and its relation to cell division in an organism such as S. pombe, whose chromosomes are relatively large, few in number, condense, and are visible by light microscopy, and whose centromere regions are moderately complex, offers an approach to understanding chromosome segregation mechanisms as they function both normally and abnormally in higher eukaryotic organisms.